The invention relates generally to the fabrication and use of a microfluidic flow cell subassembly for encapsulating a sample to allow for subsequent controlled delivery of reagents to the sample, such as multiplexed in situ biomarker staining and analysis of a mounted biological sample using dye cycling.
For multiplexed in situ biomarker analysis, tissue samples or tissue microarrays (TMA) mounted on glass slides need to be stained with multiple molecular probes to investigate biomarker expression or spatial distribution quantitatively or qualitatively. The staining and data collection processes are typically performed using time-consuming manual techniques that are susceptible to error. After staining, a coverslip must be placed over the sample in order to keep the sample wet during subsequent imaging (data collection). The coverslip must then be removed before the next round of staining. This process of cover slipping and de-cover slipping can result in loss of the sample or movement of the sample on the glass slide, which confounds downstream analysis. Staining is generally conducted by applying the staining reagent to the sample and letting it sit over the course of a pre-determined incubation. Thus, the staining time is dictated by molecular diffusion of the staining constituents from the bulk solution to the sample. Methods of actively mixing reagents on top of the sample during the incubation aim to ensure uniform staining across the sample and increase interaction between the staining constituents and the sample. However, such methods have a lower limit on reagent volume since they rely on inducing bulk fluid movement without areas of fluid separation that would affect staining uniformity.
Thus, a need exists for a system that can automate the in situ multiplexed biomarker analysis workflow while providing optimal conditions for reagent delivery and data collection. One way to control reagent delivery with small reagent volumes is to confine the reagents to an area close to the sample by using a fluidic channel. The diffusion length is determined by the height the channel, and fresh (well-mixed) reagents can be flowed through the channel to maintain the optimal reagent concentration near the sample.
In general microfluidic flow cells are comprised of one or more sealing layers sandwiched between two substantially flat substrate layers. The sealing layer creates the fluidic channel shape, forms one portion of the channel wall, and typically defines the channel thickness. This sealing layer can be formed by cutting or molding a defined shape out of a solid material, or by printing a liquid material that solidifies on one of the substrates. The two substrates enclose the fluidic channel and serve as the top and bottom channel walls. A leak-proof seal is made by clamping the sealing layer in between the substrates and/or adhering the layer to one or both of the substrates.
Furthermore, construction of the flow cell dictates that the fluidic interfaces, inlets and outlets, are formed in at least one of the substrate layers. This limits the choice of substrate materials since holes must be created through the entire substrate thickness without affecting the structural robustness. For instance, drilling holes in a glass coverslip is a time-consuming, costly process since care must be taken to avoid introducing weak points that may propagate cracks.
Thus, a microfluidic flow cell is needed that allows for a wide range of substrate materials and does not require fluidic connections to be made through any of the substrates.